JP4941672B2 - Nonspecific adsorption preventive coating agent for biological substances - Google Patents

Description

  The present invention, for example, prevents nonspecific adsorption of biologically related substances such as various proteins to parts, instruments and containers used in clinical diagnosis, biochemical experiments, etc. The present invention relates to a specific adsorption preventing coating agent.

  In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. Even for diagnostic agents using polystyrene plates and magnetic particles, for the purpose of improving sensitivity, the method that uses enzyme color development as a detection method is being switched to a method that uses fluorescence or chemiluminescence, which provides higher sensitivity. Is not sufficiently sensitive. The reason for this is that in the diagnosis of detecting specific substances in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. adsorb nonspecifically to instruments and containers, resulting in noise. This is because it increases and prevents high sensitivity. For this reason, in immunodiagnostic measurement, biological substances such as albumin, casein, and gelatin are usually used as additives in order to reduce the decrease in sensitivity due to nonspecific adsorption of these substances to instruments and containers. Thus, non-specific adsorption is suppressed and noise is reduced. However, even when these additives are used, non-specific adsorption still remains, and when biologically derived additives are used, there is a problem of biological contamination represented by BSE. In addition, the nonspecific adsorption preventing effect of these additives is easily reduced by washing with an acid / alkali, aqueous surfactant solution, or the like, resulting in poor durability.

Examples of a coating agent made of a chemically synthesized polymer for the purpose of preventing nonspecific adsorption include polyoxyethylene in JP-A-5-103831, JP-A-2006-177914, and JP-A-2006-299045. And copolymers having phosphorylcholine groups have been proposed in JP-A-2006-176720 and JP-A-2006-258585, but these non-specific adsorption preventing effects were insufficient. .
JP-A-5-103831 JP 2006-177914 A JP 2006-299045 A JP 2006-176720 A JP 2006-258585 A

  The present invention is capable of preventing non-specific adsorption of biologically related substances such as proteins to parts, instruments and containers used in clinical tests and biochemical experiments (for example, chemiluminescence immunodiagnostic measurement) and is durable. Provided is a non-specific adsorption-preventing coating agent for a biologically relevant substance having high properties.

  In order to solve this problem, the present inventors have synthesized a copolymer having a specific composition using a specific cross-linking agent, thereby achieving a high non-specific adsorption preventing effect and durability on a biological substance. It was found that a compatible coating agent was obtained, and the present invention was completed.

The non-specific adsorption-preventing coating agent for biologically related substances according to one aspect of the present invention is
A water-soluble copolymer (P) having a repeating unit (A) represented by the following general formula (1) and a repeating unit (B) represented by the following general formula (2);
A hydrazide compound (H) having at least two hydrazino groups per molecule;
Containing.

・・・・・（１）
（式中、Ｒ^０は水素原子またはメチル基を表し、Ｘは下記式（１ａ）または（１ｂ）で表される基を示す。）

(1)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and X represents a group represented by the following formula (1a) or (1b)).

・・・・・（１ａ）
（式中、Ｒ^１およびＲ^２は独立して、水素原子、炭素数１〜８のアルキル基、或いは、水酸基、カルボキシル基、アルコキシ基、アシロキシ基、およびアルコキシカルボニル基から選ばれる少なくとも１つの基で置換された炭素数１〜８のアルキル基である。）

(1a)
Wherein R ¹ and R ² are independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or at least one group selected from a hydroxyl group, a carboxyl group, an alkoxy group, an acyloxy group, and an alkoxycarbonyl group. And an alkyl group having 1 to 8 carbon atoms substituted with

・・・・・（１ｂ）
（式中、Ｒ^３およびＲ^４は独立して、単結合、置換または非置換のメチレン基、或いは、置換または非置換の炭素数２〜７の２価のアルキレン基（ここで、Ｒ^３およびＲ^４の炭素数の合計は４〜１０である。）を示し、Ｙは単結合、Ｏ、およびＳの何れかを示す。）

(1b)
(Wherein R ³ and R ⁴ are independently a single bond, a substituted or unsubstituted methylene group, or a substituted or unsubstituted divalent alkylene group having 2 to 7 carbon atoms (wherein R ³ and R ^{4 are} The total number of carbon atoms of R ⁴ is 4 to 10.), and Y represents a single bond, O, or S.)

・・・・・（２）
（式中、Ｒ^５は水素原子またはメチル基を示し、Ｚは少なくとも１個のアルド基またはケト基を有する有機基を示す。）
なお、本発明において、生体関連物質とは脂質、タンパク質、糖類、または核酸類をいう。

(2)
(In the formula, R ⁵ represents a hydrogen atom or a methyl group, and Z represents an organic group having at least one aldo group or keto group.)
In the present invention, the biological substance means a lipid, protein, saccharide, or nucleic acid.

  In the non-specific adsorption preventing coating agent, the water-soluble copolymer (P) may further have a repeating unit (C) represented by the following general formula (3).

・・・・・（３）
（式中、Ｒ^６は水素原子またはメチル基を示す。）

(3)
(In the formula, R ⁶ represents a hydrogen atom or a methyl group.)

  According to the non-specific adsorption-preventing coating agent for a biological substance, the water-soluble copolymer (P) having the repeating unit (A) and the repeating unit (B) and the hydrazide compound (H) are contained. As a result, a coating layer having a high non-specific adsorption preventing effect and durability can be formed.

  The coating agent for nonspecific adsorption of a biological substance according to one embodiment of the present invention includes a repeating unit (A) represented by the following general formula (1) and a repeating unit (B represented by the following general formula (2): And a hydrazide compound (H) having at least two hydrazino groups per molecule.

・・・・・（１）
（式中、Ｒ^０は水素原子またはメチル基を表し、Ｘは下記式（１ａ）または（１ｂ）で表される基を示す。）

(1)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and X represents a group represented by the following formula (1a) or (1b)).

・・・・・（１ａ）
（式中、Ｒ^１およびＲ^２は独立して、水素原子、炭素数１〜８のアルキル基、或いは、水酸基、カルボキシル基、アルコキシ基、アシロキシ基、およびアルコキシカルボニル基から選ばれる少なくとも１つの基で置換された炭素数１〜８のアルキル基である。）

(1a)
Wherein R ¹ and R ² are independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or at least one group selected from a hydroxyl group, a carboxyl group, an alkoxy group, an acyloxy group, and an alkoxycarbonyl group. And an alkyl group having 1 to 8 carbon atoms substituted with

・・・・・（１ｂ）
（式中、Ｒ^３およびＲ^４は独立して、単結合、置換または非置換のメチレン基、或いは、置換または非置換の炭素数２〜７の２価のアルキレン基（ここで、Ｒ^３およびＲ^４の炭素数の合計は４〜１０である。）を示し、Ｙは単結合、Ｏ、およびＳの何れかを示す。）

(1b)
(Wherein R ³ and R ⁴ are independently a single bond, a substituted or unsubstituted methylene group, or a substituted or unsubstituted divalent alkylene group having 2 to 7 carbon atoms (wherein R ³ and R ^{4 are} The total number of carbon atoms of R ⁴ is 4 to 10.), and Y represents a single bond, O, or S.)

・・・・・（２）
（式中、Ｒ^５は水素原子またはメチル基を示し、Ｚは少なくとも１個のアルド基またはケト基を有する有機基を示す。）

(2)
(In the formula, R ⁵ represents a hydrogen atom or a methyl group, and Z represents an organic group having at least one aldo group or keto group.)

1. Water-soluble copolymer (P)
1.1. Physical Properties / Structure of Water-Soluble Copolymer (P) The weight average molecular weight of the water-soluble copolymer (P) contained in the non-specific adsorption-preventing coating agent for biologically related substances according to this embodiment is usually 1,000 to 1,000,000, preferably 2,000 to 100,000, and more preferably 3,000 to 50,000. The molecular weight distribution of the water-soluble copolymer (P) typically has a weight average molecular weight / number average molecular weight of 1.5 to 3. If the weight average molecular weight of the water-soluble copolymer (P) is less than 1,000, the nonspecific adsorption preventing effect may be insufficient, while the water-soluble copolymer (P) has a weight average molecular weight of If it is greater than 1,000,000, the viscosity of the solution may increase and coating may become difficult.

  The water-soluble copolymer (P) is water-soluble. In the present invention, “water-soluble” means that when a copolymer is added to and mixed with water so as to have a polymer solid content of 1% at 25 ° C., it is visually or translucently dissolved. Say.

1.1.1. Repeating unit (A)
In the water-soluble copolymer (P), the repeating unit (A) represented by the general formula (1) has a high non-specific adsorption preventing effect and the solubility of the water-soluble copolymer (P) in water. Contribute to.

In the general formula (1a), R ¹ is preferably at least one selected from a hydrogen atom, a methyl group and an ethyl group, and R ² is preferably a hydrogen atom, a methyl group, an ethyl group, a normal propyl group, an isopropyl group. , At least one selected from a methoxyethyl group and a hydroxyethyl group. The most preferred combination is that R ¹ and R ² are both methyl groups in that they are excellent in the effect of preventing non-specific adsorption and can provide moderate solubility in water.

In the general formula (1b), R ³ and R ⁴ are preferably a methylene group, an ethylene group, or a propylene group, and Y is preferably a single bond or an oxygen atom. The most preferable combination is that R ³ and R ⁴ are both ethylene groups, and Y is an oxygen atom in that it has an excellent non-specific adsorption preventing effect and can provide moderate solubility in water.

1.1.2. Repeating unit (B)
In the water-soluble copolymer (P), the repeating unit (B) represented by the general formula (2) contributes to the durability of the coat layer.

In the general formula (2), R ⁵ is preferably a hydrogen atom. Z is preferably an aldo group such as a formyl group, a formylphenyl group, an ester group containing an aldo group represented by the following general formula (4), or an amide group containing an aldo group represented by the following general formula (5). Having an organic group;

・・・・・（４）
（式中、Ｒ^７〜Ｒ^１０は独立して、水素原子、メチル基、またはエチル基である。）

(4)
(In formula, R < ⁷ > -R < ¹⁰ > is a hydrogen atom, a methyl group, or an ethyl group independently.)

・・・・・（５）
（式中、Ｒ^１１〜Ｒ^１４は独立して、水素原子、メチル基、またはエチル基である。）
アセチル基、アセチルフェニル基、下記一般式（６）で示されるケト基を含有するエステル基、下記一般式（７）で示されるケト基を含有するアミド基などのケト基を有する有機基である。

(5)
(Wherein R ^{11 to} R ¹⁴ are each independently a hydrogen atom, a methyl group, or an ethyl group.)
An organic group having a keto group such as an acetyl group, an acetylphenyl group, an ester group containing a keto group represented by the following general formula (6), an amide group containing a keto group represented by the following general formula (7) .

・・・・・（６）
（式中、Ｒ^１５〜Ｒ^１８は独立して、水素原子、メチル基、またはエチル基である。）

(6)
(Wherein R ^{15 to} R ¹⁸ are independently a hydrogen atom, a methyl group, or an ethyl group.)

・・・・・（７）
（式中、Ｒ^１９〜Ｒ^２２は独立して、水素原子、メチル基、またはエチル基である。）
Ｚとして、より好ましい有機基は、ホルミル基、アセチル基、および下記式（８）で示される有機基であり、最も好ましい有機基は、下記式（８）で示される有機基である。

(7)
(In the formula, R ^{19 to} R ²² are each independently a hydrogen atom, a methyl group, or an ethyl group.)
As Z, more preferred organic groups are a formyl group, an acetyl group, and an organic group represented by the following formula (8), and the most preferred organic group is an organic group represented by the following formula (8).

・・・・・（８）
なお、本発明において、アルド基とは炭素原子に結合したアルデヒドを意味し、ケト基とは２個の炭素原子と結合したカルボニル基を意味し、カルボキシル基やアミド基は含まない。

(8)
In the present invention, the aldo group means an aldehyde bonded to a carbon atom, the keto group means a carbonyl group bonded to two carbon atoms, and does not include a carboxyl group or an amide group.

1.1.3. Repeating unit (C)
The water-soluble copolymer (P) may further have a repeating unit (C) represented by the following general formula (3). In the water-soluble copolymer (P), the repeating unit (C) represented by the general formula (3) contributes to the development of a high non-specific adsorption preventing effect and the adsorption of the coat layer to the substrate.

・・・・・（３）
（式中、Ｒ^６は水素原子またはメチル基を示す。）
上記一般式（３）において、Ｒ^６は好ましくは水素原子である。

(3)
(In the formula, R ⁶ represents a hydrogen atom or a methyl group.)
In the above general formula (3), R ⁶ is preferably a hydrogen atom.

1.1.4. Repeating unit (D)
In the water-soluble copolymer (P), a repeating unit (D) other than the repeating units (A) to (C) represented by the general formulas (1) to (3) as long as the effects of the present invention are not impaired. May be included.

1.2. Manufacture of water-soluble copolymer (P) Next, the monomer composition used in order to manufacture water-soluble copolymer (P) is demonstrated.

1.2.1. Monomer (a)
The repeating unit (A) has a structure derived from at least one monomer (a). The monomer (a) is preferably at least one monomer selected from acrylamide and N-substituted monomers of acrylamide. More preferably, the monomer (a) is at least one selected from acrylamide, N-hydroxyethylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N, N-diethylacrylamide, and acryloylmorpholine. Seed, more preferably N, N-dimethylacrylamide. The amount of the monomer (a) used is preferably 20 to 99 parts by weight, more preferably 25 to 98 parts by weight, further preferably 100 parts by weight of the total amount of monomers for producing the water-soluble copolymer (P). Is 30 to 95 parts by weight. Here, when the weight part of the monomer (a) is less than 20 parts by weight per 100 parts by weight of the total amount of monomers for producing the water-soluble copolymer (P), the film formation is poor or non-specific adsorption prevention The effect may be inferior, whereas if it exceeds 99 parts by weight, the durability may be poor.

1.2.2. Monomer (b)
The repeating unit (B) has a structure derived from at least one monomer (b). The monomer (b) is preferably at least one selected from acrolein, formylstyrene, vinyl methyl ketone, vinyl phenyl ketone, (meth) acrylates represented by the above formulas (4) to (7), and (meth) acrylamides. More preferably at least one selected from acrolein, vinyl methyl ketone, and diacetone acrylamide, and most preferably diacetone acrylamide from the viewpoint of monomer safety. is there. The amount of the monomer (b) used is preferably 1 to 80 parts by weight, more preferably 2 to 75 parts by weight, further preferably 100 parts by weight of the total amount of monomers for producing the water-soluble copolymer (P). Is 5 to 70 parts by weight. Here, when the weight part of the monomer (b) is less than 1 part by weight per 100 parts by weight of the total amount of monomers for producing the water-soluble copolymer (P), durability may be deteriorated, If it exceeds 80 parts by weight, the film formation may be poor or the non-specific adsorption preventing effect may be poor.

1.2.3. Monomer (c)
The repeating unit (C) has a structure derived from at least one monomer (c). The monomer (c) is preferably at least one selected from 2-methoxyethyl acrylate and 2-methoxyethyl methacrylate, more preferably 2-methoxyethyl acrylate. The amount of the monomer (c) used is preferably 2 to 50 parts by weight, more preferably 20 to 48 parts by weight, and still more preferably with respect to 100 parts by weight of the total amount of monomers for producing the water-soluble copolymer (P). Is 30 to 45 parts by weight. Here, when the weight part of the monomer (c) is within the above range, high durability and good film forming property can be expected.

  The water-soluble copolymer according to the present embodiment may contain a repeating unit (D) in addition to the repeating units (A) to (C). The monomer (d) is a component for forming the repeating unit (D). As other monomers (d), anionic monomers, in particular, styrene sulfonic acid, isoprene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, etc. are copolymerized with monomers (a) to (c). By producing a coalescence, a high low non-specific adsorption effect may be obtained when used as a coating agent such as a blood bag. As other monomer (d), N other than methyl methacrylate, ethyl acrylate, butyl acrylate, styrene, polyoxyethylene (meth) acrylic ester, monomer (a) for film forming property, water resistance, moisture retention and the like control -Known monomers such as substituted acrylamides may be used.

  The monomer to be used can be used for copolymerization after being purified as an industrial raw material or unpurified.

  The polymerization of the monomer can be performed by a known polymerization method such as radical polymerization, anionic polymerization, or cationic polymerization, and is preferably radical polymerization because of easy production.

  The polymerization of the monomer is carried out by stirring and heating together with a known solvent, initiator, chain transfer agent and the like. The polymerization time is usually from 30 minutes to 24 hours, and the polymerization temperature is usually from about 0 to 120 ° C.

  The water-soluble copolymer (P) after polymerization is preferably purified by reprecipitation, extraction, dialysis or the like.

2. Hydrazide compound (H)
The hydrazide compound (H) has at least two hydrazino groups in one molecule. Examples of such hydrazide compounds (H) include oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, sebacic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, Dicarboxylic acid dihydrazides having 2 to 10 carbon atoms, particularly 4 to 6 carbon atoms, such as acid dihydrazide, fumaric acid dihydrazide, itaconic acid dihydrazide; citric acid trihydrazide, nitriloacetic acid trihydrazide, cyclohexanetricarboxylic acid trihydrazide, ethylenediaminetetraacetic acid tetraacetate Trifunctional or higher hydrazides such as hydrazide; ethylene-1,2-dihydrazine, propylene-1,2-dihydrazine, propylene-1,3-dihydrazine, butylene -1,2-dihydrazine, butylene-1,3-dihydrazine, butylene-1,4-dihydrazine, butylene-2,3-dihydrazine, etc. aliphatic dihydrazines having 2 to 4 carbon atoms in total, etc. Water-soluble dihydrazine and at least some hydrazino groups of these polyfunctional hydrazine derivatives are converted to acetaldehyde, propionaldehyde, butyraldehyde, acetone, methyl ethyl ketone, diethyl ketone, methyl-n-propyl ketone, methyl-n-butyl ketone. Examples thereof include compounds blocked by reacting with a carbonyl compound such as diacetone alcohol, such as adipic acid dihydrazide monoacetone hydrazone, adipic acid dihydrazide diacetone hydrazone, and the like. Among these hydrazide compounds (H), at least one selected from adipic acid dihydrazide, sebacic acid dihydrazide, isophthalic acid dihydrazide, and adipic acid dihydrazide diacetone hydrazone is preferable. A hydrazide compound (H) can be used individually or in mixture of 2 or more types.

  The amount of the hydrazide compound (H) used is such that the molar equivalent ratio of the total amount of aldo groups and keto groups in the water-soluble copolymer (P) to the hydrazino group of the hydrazide compound (H) is 1: 0.1. 5, preferably in an amount ranging from 1: 0.5 to 1.5, more preferably from 1: 0.7 to 1.2. In this case, if the hydrazino group is less than 0.1 equivalent with respect to the total of 1 equivalent of the aldo group and keto group, the durability of the coating layer may be reduced. The adsorption preventing effect may be reduced.

  The hydrazide compound (H) can be blended in an appropriate step for preparing the non-specific adsorption-preventing coating agent for biologically relevant substances according to this embodiment, but the polymerization stability during the production of the water-soluble copolymer (P). In order to maintain the properties, it is preferable to add the total amount of the hydrazide compound (H) after the production of the water-soluble copolymer (P).

  The hydrazide compound (H) is a keto group and / or aldo group of the water-soluble copolymer (P) in the drying process after application of the non-specific adsorption-preventing coating agent for the biological substance according to this embodiment. It reacts with to form a hydrophilic network structure and has a function of crosslinking the coating layer. This crosslinking reaction usually proceeds at room temperature without using a catalyst, but if necessary, a catalyst such as a water-soluble metal salt such as zinc sulfate, manganese sulfate, cobalt sulfate or the like is added, or it is heated and dried. Can be promoted.

3. Non-specific adsorption preventing coating agent The non-specific adsorption preventing coating agent for a biological substance according to this embodiment preferably contains a solvent in addition to the water-soluble copolymer (P) and the hydrazide compound (H). The solvent is preferably water. The solid content concentration of the non-specific adsorption preventing coating agent is preferably 0.01% to 30%, more preferably 0.1% to 10%.

  The non-specific adsorption preventing coating agent for a biological substance according to this embodiment has a high non-specific adsorption preventing effect and durability. Specifically, the non-specific adsorption-preventing coating agent for biologically related substances according to this embodiment can act as follows.

  That is, in the non-specific adsorption-preventing coating agent for bio-related substances according to the present embodiment, the water-soluble copolymer (P) is adsorbed on the wall surface of the container / equipment, etc. by the hydrophobic bond of the repeating unit (B), Preventing nonspecific adsorption of proteins and the like by making the wall surface hydrophilic with the repeating unit (A) (when the water-soluble copolymer (P) contains the repeating unit (C), the repeating unit (C)). Can do.

  Further, in the non-specific adsorption preventing coating agent for a biological substance according to the present embodiment, the hydrazino group in the hydrazide compound (H) is a keto group in the repeating unit (B) of the water-soluble copolymer (P) and / or Alternatively, it can react with an aldo group to form a hydrophilic network structure, crosslink the coat layer, and develop the durability of the coat layer.

  For example, the coating agent for nonspecific adsorption of a biological substance according to the present embodiment is used by coating the surface of a part, container, or instrument used in clinical diagnosis, biochemical experiment, etc. (for example, chemiluminescence immunodiagnostic measurement). Is done. The coating can be carried out by a known method. For example, spin coating, bar coating, blade coating, curtain coating, spray coating, dip coating and the like are possible. Dip coating is preferable because it can be applied to a device having a complicated shape without a defect. After coating, it is preferable to dry at normal temperature or heat. Although preferable heating temperature and time are based on the base and the like, they are about 40 to 120 ° C. and about 3 minutes to 24 hours.

  Examples of the non-specific adsorption preventing coating agent for biologically related substances according to this embodiment include clinical diagnostic parts such as optical cells, specimen transfer tubes, specimen tubes, etc .; silicon base materials represented by MEMS, and glass represented by microfluidics Non-specific adsorption of various proteins, lipids, nucleic acids, etc. can be suitably prevented by coating biochips such as substrates and plastic substrates; biosensors such as gold-deposited substrates. A coating layer exhibiting durability can be formed.

4). Examples and Comparative Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

  In this example, the weight average molecular weight (Mw) is a TSKgel α-M column manufactured by Tosoh Corporation, the flow rate is 1 ml / min, the elution solvent is a 0.1 mM sodium chloride aqueous solution / ethanol mixed solvent, the column temperature is 40 ° C. The measurement was performed by gel permeation chromatography (GPC) using monodisperse polyethylene glycol as a standard. Absorbance was measured with a model 680 microplate reader manufactured by Nippon Bio-Rad Laboratories.

4.1. Example 1
4.1.1. Synthesis of water-soluble copolymer (P-1) 40 g of dimethylacrylamide as monomer (a), 20 g of diacetone acrylamide as monomer (b), 40 g of methoxyethyl acrylate as monomer (c), and mercaptopropionic acid as chain transfer agent 1 g was mixed with 900 g of water and placed in a separable flask equipped with a stirrer. The temperature was raised to 70 ° C. while blowing nitrogen, and after adding 4 g of 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride as an initiator, polymerization was continued for 2 hours. The mixture was further heated to 80 ° C. and aged for 3 hours, and then cooled to room temperature. The obtained copolymer solution was purified by a dialyzer and further diluted with water to obtain 1800 g of a 5% water-soluble copolymer (P-1) aqueous solution of this example. The weight average molecular weight by GPC of the water-soluble copolymer (P-1) was 27,000.

4.1.2. Coating The 5% water-soluble copolymer (P-1) aqueous solution was further diluted to 1% with water, and 9 g of adipic acid dihydrazide was added and dissolved as a hydrazide compound (H) to prepare a coating agent. A 96-well plate made of polystyrene (hereinafter referred to as “96-well plate”) was dipped in this coating agent, pulled up, washed with replacement water, and the remaining water was blown off with an air gun. This was dried at 40 ° C. for 3 hours to obtain a coated 96-well plate.

4.1.3. Measurement of non-specific adsorption prevention effect A coated 96-well plate was washed three times with water, filled with an aqueous solution of horseradish peroxidase-labeled mouse IgG antibody ("AP124P" manufactured by Millipore), and incubated at room temperature for 30 minutes. Thereafter, the plate was washed three times with PBS buffer, and developed with TMB (3,3 ′, 5,5′-tetramethylbenzidine) / hydrogen peroxide solution / sulfuric acid, and the absorbance at 450 nm was measured.

4.1.4. Measurement of durability The coated 96-well plate was washed 5 times with a 2% aqueous sodium dodecyl sulfate solution, and the effect of preventing nonspecific adsorption was measured.

4.2. Examples 2-4
The same operation as in Example 1 was performed except that the monomer and the hydrazide compound (H) had the compositions shown in Table 1.

4.3. Comparative Example 1
In Example 1, the same operation as in Example 1 was performed except that the hydrazide compound (H) was not used.

4.4. Comparative Examples 2 and 3
The same operation as in Example 1 was performed except that the monomer composition shown in Table 1 was used. However, when polymerized with the monomer composition of Comparative Example 3, polymer solids were precipitated during the polymerization, and stirring became difficult, so the polymerization was stopped and left unrated.

4.5. Comparative Example 4
In Example 1, using bovine serum albumin (BSA) instead of the coating agent, the absorbance in the case of using BSA in the same manner as in “4.1.3. Was measured.

4.6. Measurement Results Table 1 shows the measurement results of the nonspecific adsorption preventing effect in the above Examples and Comparative Examples.

  According to Table 1, by using the non-specific adsorption-preventing coating agent for biologically relevant substances according to the present embodiment, when the hydrazide compound (H) is not used, the co-polymer synthesized without using the monomer (c). When combined, compared to the case of using bovine serum albumin, the nonspecific adsorption amount of mouse IgG antibody to the 96-well plate can be remarkably reduced, and the formed coating layer is confirmed to be excellent in durability. It was done.

Claims (2)

A water-soluble copolymer (P) having a repeating unit (A) represented by the following general formula (1) and a repeating unit (B) represented by the following general formula (2);
A hydrazide compound (H) having at least two hydrazino groups per molecule;
A non-specific adsorption-preventing coating agent for bio-related substances, comprising:

(1)
(In the formula, R ⁰ represents a hydrogen atom or a methyl group, and X represents a group represented by the following formula (1a) or (1b)).

(1a)
Wherein R ¹ and R ² are independently a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or at least one group selected from a hydroxyl group, a carboxyl group, an alkoxy group, an acyloxy group, and an alkoxycarbonyl group. And an alkyl group having 1 to 8 carbon atoms substituted with

(1b)
(Wherein R ³ and R ⁴ are independently a single bond, a substituted or unsubstituted methylene group, or a substituted or unsubstituted divalent alkylene group having 2 to 7 carbon atoms (wherein R ³ and R ^{4 are} The total number of carbon atoms of R ⁴ is 4 to 10.), and Y represents a single bond, O, or S.)

(2)
(In the formula, R ⁵ represents a hydrogen atom or a methyl group, and Z represents an organic group having at least one aldo group or keto group.) The non-specific adsorption-preventing coating agent for a biological substance according to claim 1, wherein the water-soluble copolymer (P) further has a repeating unit (C) represented by the following general formula (3).

(3)
(In the formula, R ⁶ represents a hydrogen atom or a methyl group.)